A组轮状病毒一步法RT-PCR检测技术的建立和应用研究  

The application research of detective techniqne of a single step RT-PCR to A Group rotavirus

作　　者：王信隆[1] 闫小君[2] 梁未丽[1] 王莹[3] 

机构地区：[1]西安交通大学医学院微生物学教研室,陕西西安710061 [2]第四军医大学基因诊断研究所 [3]西安交通大学医学院生理学教研室,陕西西安710061

出　　处：《细胞与分子免疫学杂志》2001年第5期450-451,共2页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的设计A组轮状病毒RV特异性引物,应用Tth酶,建立一步法RT-PCR扩增方案,检测西安地区腹泻幼儿196份粪便标本,并与本室研制建立的反向间接血凝法RPHA,聚丙烯酰胺凝胶电泳,市售ELISA试剂盒平行检测对比分析。方法设计并合成第九基因序列保守区互补的特异性引物,在经典法RT-PCR试验成功的基础上,建立合理的一步法RT-PCR。结果四种检测结果阳性率分别为33.16%,23.47%,21.94%和25%,X2检验表明,RT-PCR最为敏感。结论反转录和PCR由Tth酶一步完成,克服了经典法中AMV、RNasin等试剂昂贵,操作繁锁等缺点,很适合临检需要。Aim To design a pair of rotavirus -specific primers of A Group,and develop amplification sc heme of a single step RT -PCR by using Tth enzyme.196samples of severe viral diarrhea were examined in infants and young children in Xi ' an.We made parallel detections on an d compared analysis with reverse passive hemagglutination(RPHA)which developed by ourselves,polyacryla mide gel electrophoresis(PAGE),and ELISA.Methods To design and synthesiz a pair of specific primers complementary to conserved sequences of the gene 9in A Group ro-tavirus.On the basis of successful c lassic RT -PCR,we developed a single step RT -PCR which is sensitiv e,specific,simple and suitable for clinical application.Results The respective positive rate of the f our methods:33.16%,23.47%,21.94%,25%,PCR proved to be the most sensitive technique in X2test.Conclusion RT and PCR can be accomplished by using Tth enzyme onl y in one step.It can overcome some disadvantages in classical methods,such as the expensiveness of the reagents(AMV,RNasin)and the complicated procedure.It' s fit for the demands of clinical examine.

关 键 词：轮状病毒 逆转录一聚合酶链反应 反向间接血凝试验 

分 类 号：R373.25[医药卫生—病原生物学]